Recently, biosensors employing enzymes have been applied to the detection of both glucose and cholesterol concentrations in blood samples. In European Patent Application 0 230 472 to Nankai et al., a biosensing instrument is disclosed which employs amperometric measurements to determine glucose concentration in a blood sample. The instrument employs a test cell with measuring, reference and counter electrodes. Overlaying the electrodes is an insert which contains glucose oxidase, potassium ferricyanide and other components. When a blood sample is placed in contact with the insert, glucose in the sample reacts with the potassium ferricyanide (through the action of the glucose oxidase) to form potassium ferrocyanide. A subsequent application of a voltage to the electrodes induces a reversal of the reaction and a current flow which is proportional to the concentration of the potassium ferrocyanide formed in the initial reaction. A measure of the current flow is said to correspond to the concentration of glucose in the sample.
A similar system for measuring both glucose and cholesterol concentrations is disclosed in PCT International Application No. WO 89/08713 of Pottgen et al. Both the Nankai et al. and the Pottgen et al. systems employ similar chemistries to enable amperometric detection of glucose concentrations. For glucose, both rely upon the catalytic action of glucose oxidase on glucose to enable the conversion of potassium ferricyanide (+3) to potassium ferrocyanide (+4), (i.e., the "forward" reaction). A subsequent application of a potential across the reactants electrochemically causes a reversal of the reaction, (i.e. the "reverse" reaction). Upon the oxidation of glucose by glucose oxidase, electrons are transferred to ferricyanide causing its reduction yielding ferrocyanide. An applied potential to the electrode electrochemically oxidizes ferrocyanide back to ferricyanide with the electrons transferred to the electrode. This creates a small and detectable electrical current whose level is proportional to the level of glucose concentration in the sample. The current which results during the reverse reaction is known as the Cottrell current and is described by the following equation: ##EQU1## where: n=the number of transferred electrons;
F=Faraday's constant PA1 A=area of measuring electrode; PA1 C=concentration of the analyte; PA1 D=diffusion coefficient of the electroactive species; PA1 t=time
Equation 1 can be reduced to a simpler expression by realizing that most of the factors in the equation are constants for any particular test system. Thus, the Cottrell current, at any particular time during the reverse reaction, is shown by the following: ##EQU2##
Equation 2 indicates that the Cottrell current is proportional to the concentration of the analyte and is inversely proportional to the square root of the measurement time. Plots of Cottrell current variations at various glucose concentration levels, are shown by the curves in the right upper quadrant of FIG. 3.
The prior art has characteristically selected a particular time during the reverse reaction to obtain a reading of the Cottrell current and converted that reading into a measure of glucose or cholesterol concentration. Neither Nankai et al. or Pottgen et al. deal with certain real-life problems which occur during the use of a test cell. For instance, if the blood sample does not totally cover the sensing electrode surfaces, an erroneous reading results. Furthermore, if the reaction area becomes hydrated, either prior to or during the test, an erroneous reading occurs. Likewise, if there is leakage along the length of the electrodes so that the blood sample covers not only the portion of the electrodes in the reaction zone, but also outside of the reaction zone, again, erroneous readings will occur. These errors appear as baseline shifts in the Cottrell current or modulations of area during the measurement period.
Accordingly, it is an object of this invention to provide an amperometric biosensor and method which both provides analyte concentration readings and prevents erroneous readings from being reported as true.
It is another object of this invention to provide an amperometric biosensor and method for glucose concentration which provides an error indication, if an aberrant current curve results.